title: Polymorphism in ftsI gene and {beta}-lactam susceptibility in Portuguese Haemophilus influenzae strains: clonal dissemination of beta-lactamase-positive isolates with decreased susceptibility to amoxicillin/clavulanic acid.
abstract: OBJECTIVE
  The aim of this study was to characterize ampicillin resistance mechanisms in clinical isolates of Haemophilus influenzae from Portugal. Association between specific patterns of amino acid substitutions in penicillin-binding protein 3 (PBP3) (with or without β-lactamase production) and β-lactam susceptibility as well as genetic relatedness among isolates were investigated.
  
  
  METHODS
  Two-hundred and forty non-consecutive H. influenzae isolates chosen according to their different ampicillin MICs [101 β-lactamase-non-producing ampicillin-resistant (BLNAR) isolates, 80 β-lactamase-producing ampicillin-resistant (BLPAR) isolates and 59 β-lactamase-non-producing ampicillin-susceptible (BLNAS) isolates] were analysed. The β-lactamase-encoding bla(TEM-1) gene was detected by PCR. The ftsI gene encoding PBP3 was sequenced. Genetic relatedness among isolates was examined by PFGE.
  
  
  RESULTS
  Of the 240 H. influenzae isolates, 141 had mutations in the transpeptidase domain of the ftsI gene, including most BLNAR strains (94/101, 93.1%) and a high percentage of BLPAR strains (47/80, 58.8%). As previously reported, the latter have been described as β-lactamase-positive amoxicillin/clavulanic acid resistant (BLPACR). The most common amino acid substitutions were identified near the KTG motif: N526K (136/141, 96.5%), V547I (124/141, 87.9%) and N569S (121/141, 85.8%). The 141 strains were divided into 31 ftsI mutation patterns and included six groups (I, IIa, IIb, IIc, IId and III-like). BLNAR strains were genetically diverse but close genetic relationships were demonstrated among BLPACR strains.
  
  
  CONCLUSIONS
  This study shows that the non-enzymatic mechanism of resistance to β-lactams is widespread among H. influenzae isolates in Portugal. Clonal dissemination of BLPACR strains showing high resistance to ampicillin and reduced susceptibility to amoxicillin/clavulanic acid was documented.
authors.affiliation: National Reference Laboratory for Bacterial Respiratory Infections, Department of Infectious Disease, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal.
authors.forename: Ana Raquel
authors.initials: AR
authors.lastname: Barbosa
authors.forename: Maria
authors.initials: M
authors.lastname: Giufrè
authors.forename: Marina
authors.initials: M
authors.lastname: Cerquetti
authors.forename: Maria Paula
authors.initials: MP
authors.lastname: Bajanca-Lavado
mesh_terms: D000293:Adolescent; D000328:Adult; D019980:Amoxicillin-Potassium Clavulanate Combination / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D002648:Child; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D005838:Genotype; D006192:Haemophilus Infections / Q000453:epidemiology* / Q000382:microbiology; D006193:Haemophilus influenzae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests; D058889:Molecular Typing; D046915:Penicillin-Binding Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011174:Portugal / Q000453:epidemiology; D017422:Sequence Analysis, DNA; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000096:biosynthesis
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D019980:Amoxicillin-Potassium Clavulanate Combination; D001618:beta-Lactamases